Stem cells are undifferentiated cells that possess two hallmark properties; self-renewal and the ability to differentiate into one or more different cell lineages. The process of self-renewal involves the self-replication of a stem cell to allow for propagation and expansion, wherein the stem cell remains in an undifferentiated state. Progenitor cells are also undifferentiated cells that have the ability to differentiate into one or more cell lineages, but have limited or no ability to self-renew. When maintained in culture, undifferentiated cells, such as stem or progenitor cells, can undergo spontaneous differentiation, thereby losing the desired, undifferentiated cell phenotype. Thus, culture methods that minimize spontaneous differentiation in order to maintain the undifferentiated stem or progenitor cell state are needed.
Keeping undifferentiated cells in an undifferentiated state is critical to their use, e.g., in industry and medicine, since a major scientific and therapeutic usefulness of these cells lies in their ability to expand into homogenous populations that can further proliferate or differentiate into mature cells as needed, e.g., for scientific study or to repair damage to cells or tissues of a patient. Once they have spontaneously differentiated in cell culture, the cells are less proliferative and less able to differentiate into different types of cells as needed. A homogenous culture of undifferentiated stem cells is therefore a highly sought after but unrealized goal of research scientists and industry.
Current methods for culturing undifferentiated cells (e.g., various types of stem cells) attempt to minimize such spontaneous differentiation by delivering fibroblast growth factor 2 (FGF2) to the cell cultures daily, or, less frequently than every day, which is known as “feeding”. FGF2 has been shown to promote self-renewal of stem cells by inhibiting differentiation of the stem cell; however this inhibition is incomplete, and the stem cell cultures tend to gradually differentiate, thereby diminishing usefulness of the stem cell culture. Furthermore, stem cells, such as embryonic or spermatogonial stem cells, typically need to be grown on mouse embryonic fibroblast (MEF) feeder cells. This is a cumbersome step that is desirable to remove and results in a population of cells stem cells contaminated with the feeder cells that cannot be used in various fertilization protocols and production of gametes.
The ability to conditionally induce the development of stem cell lines through the process of spermatogenesis in vitro for the production of gametes would provide a long-sought-after technology for biomedical research, and animal breeding particularly if such protocols could be established for a variety of species. To date, most success has been achieved in rats and mice only, leaving larger mammals such as bovine, without such advances.
The discovery that stem cells residing within fractions of dissociated mouse and rat testis cells maintain their ability to regenerate spermatogenesis in testes of recipient mice was essential to establishing such culture systems. See Brinster et al., Proc Natl Acad Sci USA 1994; 91:11303-11307; Brinster et al., Proc Natl Acad Sci USA 1994; 91:11298-11302; Clothier et al., Nature 1996; 381:418-421; Kanatsu-Shinohara et al., Biol Reprod 2003; 69:612-616; and Nagano et al., Tissue Cell 1998; 30:389-397. The ability to isolate and experimentally manipulate these stem cells has opened new doors for research on spermatozoon development, assisted reproduction, cellular therapy and genetics. See Nagano et al., Biol Reprod 1999; 60:1429-1436; Mahanoy et al., Endocrinology 2000; 141:1273-1276; Mahato et al., Mol Cell Endocrinol 2001; 178:57-63; Ogawa et al., Nat Med 2000; 6:29-34; Shinohara et al., Proc Natl Acad Sci USA 2006; 103:13624-13628; Zhang et al., J Cell Physiol 2007; 211:149-158; Kazuki et al., Gene Ther 2008; 15:617-624; Kanatsu-Shinohara et al., Cell 2004; 119:1001-1012; Kanatsu-Shinohara et al., Proc Natl Acad Sci USA 2006; 103:8018-8023; and Nagano et al., Proc Natl Acad Sci USA 2001; 98:13090-13095. In view of this potential, protocols for isolating, propagating and genetically modifying fully functional rat and mouse spermatogonial stem cells in culture have been established. See Ryu et al., Dev Biol 2004; 274:158-170; Hamra et al., Dev Biol 2004; 269:393-410; Hamra et al., Proc Natl Acad Sci USA 2002; 99:14931-14936; Hamra et al., Methods Mol Biol 2008; 450:163-179; Hamra et al., Proc Natl Acad Sci USA 2005; 102:17430-17435; Ryu et al., Proc Natl Acad Sci USA 2005; 102:14302-14307; Orwig et al., Biol Reprod 2002; 67:874-879; and Kanatsu-Shinohara et al., Biol Reprod 2008. The mouse and rat were chosen as species for these studies due to their popularity as laboratory animal models for the study of human health and disease, and due to the lack of protocols for genetically modifying the rat germline using clonally expanded stem cells from culture. See Hamra et al., Proc Natl Acad Sci USA 2002; 99:14931-14936. Considering the many potential applications of the laboratory rat as a research model, a cost-effective and easy-to-prepare culture medium was sought in this study for the derivation and continuous proliferation of primary rat spermatogonial stem cell lines in vitro.
Despite these advances, even in the rat species, the procedure remains complex and largely unsuccessful. For example, media for long-term proliferation of rodent spermatogonial stem cells in vitro are relatively complex, expensive, time-consuming to prepare, plus are most effective when applied in combination with feeder layers of fibroblasts.
As can be seen a need exists for methods of culturing spermatogonial stem cells, particularly for larger mammals such as bovines.